摘要
目的构建弓形虫核苷三磷酸水解酶-Ⅱ(NTPase-Ⅱ)基因真核表达质粒pcDNA3.1(+)-NTPase-Ⅱ并在COS-7细胞中进行瞬时表达。方法以pBAD-HisB-NTPase-Ⅱ质粒为模板,PCR扩增NTPase-Ⅱ目的基因,将其克隆到pcDNA3.1(+)真核表达载体中,双酶切及测序鉴定重组质粒。阳离子脂质体法转染COS-7细胞并经SDS-PAGE和Western Blot检测目的蛋白的表达。结果经鉴定,弓形虫pcDNA3.1(+)-NTPase-Ⅱ核酸疫苗质粒构建成功。以脂质体法转染COS-7细胞后,转染细胞可成功地表达弓形虫NTPase-Ⅱ蛋白。结论证实了弓形虫NTPase-Ⅱ蛋白能在真核细胞中表达,为该基因的核酸疫苗研究提供了实验依据。
We constructed the eukaryotic expression plasmid pcDNA3.1（＋）-NTPase-Ⅱ of Toxoplasma gondii and assess the expression of recombinant protein in COS-7cells.The prokaryotic expression plasmid pBAD-HisB-NTPase-Ⅱ constructed previously in our laboratory was used as templates to amplify the target gene by PCR.The resulting PCR products were cloned into the vector pcDNA3.1（＋）for construction of eukaryotic expression plasmid pcDNA3.1（＋）-NTPase-Ⅱ.Then,the plasmids were transfected into COS-7cells after identification with double enzyme digestion and sequencing.Finally,Western blot was performed to detect the expression of NTPase-Ⅱ in COS-7.The result revealed that Toxoplasma NTPase-Ⅱ could be expressed in COS-7cells when compared with empty pcDNA3.1（＋）control group.Taken together,the plasmid pcDNA3.1（＋）-NTPase-Ⅱ constructed successfully will play an important role in development of a potential gene vaccine.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2015年第6期519-521,526,共4页
Chinese Journal of Zoonoses
基金
浙江省科技厅公益性项目(No.2014C33161)
温州医科大学学生科研项目(No.wyx201301011)联合资助~~
关键词
刚地弓形虫
核苷三磷酸水解酶
真核表达
Toxoplasma gondii
nucleoside triphosphate hydrolase（NTPase）
eukaryotic expression
